(1) Field of the Invention
The present invention generally relates to improved enzymes for molecular biology. More specifically, mutant RNA polymerases are provided that have improved resistance to common reagents including phosphate.
(2) Description of the Related Art
RNA and DNA polymerization reactions, which result in the synthesis of RNA or DNA polynucleotides, are an integral part of a variety of techniques used in molecular biology. Such reactions include in vitro transcription and amplification techniques such as the polymerase chain reaction (PCR), RNA amplification and self-sustained sequence replication. These reactions often employ RNA polymerases, especially bacteriophage RNA polymerases such as SP6, T7 and T3, for example, in the synthesis of both labeled RNA probes and unlabeled RNA. Improved performance of the RNA polymerases utilized in these reactions would thus be beneficial.
The rate of these synthetic reactions, and the amount of product formed, is limited by several factors. Lowering the magnesium concentration and salt concentration allows the use of high concentrations of substrate nucleotides and improve the yields of a transcription reaction (U.S. Pat. Nos. 6,586,219, 6,586,218 and 5,256,555). Those techniques prevent inhibition caused by some reaction substrates, but inhibition by other substrates and reaction products, e.g., phosphate, pyrophosphate and single stranded DNA (ssDNA) can still inhibit polymerase activity.
Transcription reactions and DNA polymerase and DNA sequencing applications routinely use the enzyme inorganic pyrophosphatase since addition of that enzyme improves the yield of transcription reactions by removing pyrophosphate (Sampson & Uhlenbeck, 1988; Weitzmann et al., 1990; Cunningham & Ofengand, 1990; Tabor & Richardson, 1990). Pyrophosphatase cleaves the polymerase reaction product pyrophosphate to produce two molecules of phosphate. However, phosphate inhibits RNA polymerase, especially at high concentrations. For example, the optimal total concentration of nucleotides found by Cunningham & Ofengand (1990) of 16 mM produces 32 mM phosphate at the end of the reaction, which is inhibitory to RNA polymerase.
Although there are various protocols in molecular biology where reactions utilizing more than one enzyme are combined, the inhibition of RNA polymerases by reagents such as pyrophosphate and phosphate can thwart efforts to simplify protocols. For example, a typical protocol for amplification of mRNA involves synthesizing a first strand cDNA using reverse transcriptase, followed by a second strand cDNA synthesis using DNA polymerase, then RNA transcription from the cDNA using RNA polymerase. See, e.g., Wang et al., 2000. These protocols usually require a cDNA purification step after the second strand synthesis because buffers and reaction products present from the cDNA synthesis procedures inhibit the RNA polymerase. Some second strand synthesis buffers are available that do not have phosphate, but an RNA polymerase that is not inhibited by phosphate would make their use, or a cDNA purification step, unnecessary.
One of the characteristics of wild-type T7 RNA polymerase is the ability to carry out some level of promoter independent synthesis by using the 3′ ends of single-stranded DNA as an initiation site. For in vitro transcription reactions, substrates that give this synthesis can be DNA primers and double-stranded linearized DNA with single-stranded 3′ tails. This can especially be a problem when there are large amounts of primers present and low levels of promoter templates. This can also take place with low amounts of RNA analyte samples where single-stranded carrier DNA has been added to increase efficiency of recovery. As a consequence of this property, there can be a large amount of aberrant synthesis taking place even in the complete absence of any input RNA, thus implying that at low levels of legitimate targets, a large amount of labeled product is only contributing to background and not signal. Secondly, the formation of this promoter independent synthesis uses up reagents such that the net yield of legitimate product can be decreased by competition with the promoter independent synthesis.
A number of mutations in RNA polymerases that modify characteristics of those enzymes are known. For example, certain mutations in T7 RNA polymerase (“T7”) (e.g., Y639F/S641A; del172-173; F644Y; F667Y) allow the polymerase to utilize deoxyribonucleotides along with ribonucleotides as substrates (Kostyuk et al., 1995; Izawa et al., 1998; European Patent Application EP1403364A1). See also Joyce (1997), Izawa et al. (1998) and Brieba and Sousa (1999). Other mutations increase (e.g. K172L, Del172-173, K98R) or decrease (e.g., P266L) promoter binding strength (Tunitskaya and Lochetkov, 2002; U.S. Pat. No. 7,335,471) or alter the termination properties of the enzyme (e.g., del 163-164, R173C). See Lyakhov et al. (1992), Lyakhov et al. (1997), Tunitskaya and Lochetkov (2002). Still other mutations (e.g., N748D, N748Q, Q758C, E222K, R756M) alter promoter recognition (U.S. Pat. No. 5,385,834; Chilliserrylattil et al., 2001) or increase the thermostability of the enzyme (e.g., S430P, F849I, F880Y, S633P—U.S. Pat. No. 7,507,567). Additional T7 mutations are described in He (1996), Macdonald et al. (1994), and Yang and Richardson (1997).
Mutations analogous to some of the above mutations have been effectively made in T3 RNA polymerase (“T3”) (see, e.g., Lyakhov et al., 1997 and European Patent Application No. EP1403364), demonstrating that the various domains of these related phage RNA polymerases are functionally equivalent.
The present invention provides, in part, RNA polymerase mutants with improved characteristics, including resistance to phosphate, pyrophosphate, sodium chloride, and/or single stranded DNA that can be advantageously used in place of wild-type RNA polymerases for various molecular biology procedures.